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SUPPLEMENTARY MATERIAL
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Antioxidant and toxicological evaluation of a Tamarindus indica L. leaf
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fluid extract
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Escalona-Arranz JC*1, Perez-Rosés R2, Rodríguez-Amado J1, Morris-
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Quevedo HJ3, Mwasi LB1, Cabrera-Sotomayor O1, Machado-García R4,
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Fong-Lórez O5, Alfonso-Castillo A5 and Puente-Zapata E5.
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Pharmacy Department, Oriente University. Santiago de Cuba, Cuba
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Departament Farmacología i Química Terapéutica. Universitat de Barcelona, Spain.
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Centre for Studies in Industrial Biotechnology (CEBI). Oriente University, Santiago de
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Cuba, Cuba
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Chemistry Department, Oriente University. Santiago de Cuba, Cuba
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Medical Toxicology Centre (TOXIMED). Medical Sciences University, Santiago de
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Cuba, Cuba
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*Pharmacy Department, Oriente University. Address: Avenida Patricio Lumumba s/n,
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90500 Santiago de Cuba; Cuba. E-mail: jcea@cnt.uo.edu.cu, Phone: 0053 (22) 641411
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Fax: 0053 (22) 641411
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Abstract
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In the scientific community there is a growing interest in Tamarindus indica L leaves,
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both as a valuable nutrient and as a functional food. This paper focuses on exploring its
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safety and antioxidant properties. A tamarind leaf fluid extract (TFE) wholly
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characterized was evaluated in its anti-DPPH activity (IC50= 44.36 μg/mL) and its
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reducing power activity (IC50= 60.87 μg/mL). TFE also exhibited a high ferrous ion
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chelating capacity, with an estimated binding constant of 1.085 mol L-1 while its
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influence over nitric oxide production in human leukocytes was irregular. At low
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concentrations, TFE stimulated NO output, but it significantly inhibited it when
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concentration grew. The TFE was found to be safe at tested conditions when an acute
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oral toxicity test and an oral mucous irritability test demonstrated in both cases that it
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was a non-toxic substance. Results suggest that TFE might become a functional dietary
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supplement.
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Keywords: Tamarindus indica L., antioxidant, toxicological evaluation, functional
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foods, tamarind leaves
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1. Experimental section
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1.1. Plant material, chemicals and reagents
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Tamarind leaves were collected from a tamarind population in Santiago de Cuba,
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eastern Cuba (GPS 20º 2´38.9´´N and 075º 45´25.8´´W.), and were previously identified
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by Dr. Jorge Sierra-Calzado. A voucher specimen registered as 052216 was deposited in
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the Docent Section of The BSC Herbarium at the Biology Department of Oriente
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University. Collected leaves were sun dried (residual humidity below 10% by the stove
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method), milled (MLK, Russia), and passed across a mesh light sieve of 5 mm.
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2,2-diphenyl-1-picryl hydrazyl (DPPH), quercetin, absolute ethanol, Hanks’ balanced
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salt solution (HBSS) without Ca2+ and Mg2+, ethylenediaminetetraacetic acid
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tetrasodium salt dihydrate (EDTA-Na4.H2O), ammonium chloride (NH4Cl), potassium
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bicarbonate (KHCO3), formalin, Griess reagent (modified), L-arginine (L-Arg),
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lipopolysaccharides (LPS) from Escherichia coli 0127:B8, NG-methyl-L-arginine
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acetate salt (L-NMMA) and sodium nitrite (NaNO2) were obtained from Sigma
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Chemical Company (St. Louis, MO, USA). Potassium ferricyanide, trichloroacetic acid,
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ferric chloride and ferric sulfate were obtained from Beijing Chemical Reagents
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Company (Beijing, China), meanwhile phosphate buffer, EDTA and Tris buffer were
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supplied by Janssen Chimica (Belgium).
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1.2. Tamarind extracts preparation
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Extracting conditions were: 4 days of percolation and a mixture of ethanol/water 72%
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v:v as solvent for the procedure. Previous experiences of our research group had proved
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those conditions as a sure way to extract high quantities of metabolites (Escalona-
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Arranz et al., 2011). The tamarind fluid extract (TFE) was prepared from 4 extractions
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that were collected, mixed and concentrated up to 1 millilitre of extract for each
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milligram of dried leaves. A vacuum evaporation system at 42 ºC (KIKA WERKE,
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Germany) was used for the concentration of collected extractions. TFE had already been
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characterized in its physico-chemical and chemical properties: total soluble substances,
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pH, relative density, refraction index and total polyphenol and flavonoid content
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(Escalona-Arranz et al., 2011). Quercetin, a well known antioxidant, was identified in
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TFE and was likewise chosen as positive reference for all in vitro tests except that of
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nitric oxide production in human leukocytes.
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1.3. DPPH radical scavenging activity
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The radical scavenging activity of the extract was evaluated using 2,2-diphenyl-1-
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picrylhydrazyl (DPPH•) ( Brand-Williams et al. 1995). The fluid extract was lyophilized
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(TELSTAR, LIOALFA-6, Spain) and dissolved in absolute ethanol to prepare seven
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solutions ranging from 6.25 μg/mL until 400 μg/mL (12.5, 25, 50, 100, 200, 400).
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Quercetin was used as standard in a range of 0.78 until 50.0 μg/mL. In brief: 2 mL of
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the extract were added to 1 mL of DPPH (1 mM) in absolute ethanol. The mixture was
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incubated at room temperature in the dark for one hour. The control was prepared as
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above but without the extract. Absorbance at 517 nm was measured in a
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spectrophotometer (RAY LEIGH UV-2601, China), using absolute ethanol as blank.
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Scavenging activity was expressed as the inhibition percentage of the DPPH free stable
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radical calculated using the following equation:
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% Anti- radical activity = {(Control Abs.- Sample Abs.)/Control Abs.}x100
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Data were normalized using the hyperbolic logarithm of the concentrations tested.
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1.4. Reducing power assay
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The reducing power was determined according to the method of Oyaizu (1986). The
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fluid extract, lyophilized and dissolved in ethanol at five different concentrations (35,
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70, 140, 220 and 280 μg/mL) was tested by the reducing power assay. In brief: 1 mL of
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the extract was added to 2.5 mL of 0.2 M phosphate buffer pH=6.6 and 2.5 mL of
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potassium ferricyanide (10 mg/mL). The mixture was incubated at 50°C for 20 min.
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After incubation 2.5 mL of trichloroacetic acid (10.0 mg/mL) were added, the mixture
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was centrifuged at 1160 x g for 10 min, and then 2.5 mL of the supernatant were mixed
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with 2.5 mL of deionized water and 0.5 mL of ferric chloride (1.0 mg/mL). Five
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different solutions (from 3.75 to 60 μg/mL) of quercetin were used as positive control.
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Absorbance was then measured at 700 nm against a blank in the spectrophotometer
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(RAY LEIGH UV-2601, China). Results were expressed as % antiradical activity. In
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this experiment higher absorbance indicates higher reducing power. Data was
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normalized using the hyperbolic logarithm of tested concentrations.
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1.5. Ferrous ion chelating activity assay
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For the ferrous ion chelating activity assay the technique described by Andjelković et al.
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(2006) was followed with a minor modification. It was recorded the UV/visible
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spectrum (200-800 nm) of the TFE (five concentrations from 0.4 until 6.4 mg/mL),
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before and after adding rates of 0.01 and 0.1 mM ferric sulfate. The bathochromic shifts
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experimented for the sample under consideration was evaluated when the ferric sulfate
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was added. The wavelength in which the fluid extract-iron complex absorbs was thus
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defined. The binding constant of the fluid extract with ferrous ions is defined as k=
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intercept/slope, where intercept and slope are parameters of a linear relationship
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between 1/(concentration) and 1/(absorbance of complexed ions). A quercetin solution
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(from 0.76 until 0.228 mg/mL) was used as reference. The stability of the fluid extract-
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iron complex was determined by addition of 0.01 and 0.1 mM of EDTA. An
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absorbance’s decrease at the wavelength that absorbs the complex was considered as a
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rupture in the fluid extract-iron complex. The percentage of leftover complex was
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determined by measurement of the intensity of absorption of the complex, before and
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after EDTA addition. In order to avoid oxygen interference all stock solutions were
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freshly prepared before measuring and bi-distilled water and Tris buffer were subjected
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to sonication for 45 minutes in an ultrasonic bath. All determinations were measured in
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the spectrophotometer (RAY LEIGH UV-2601, China).
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1.6. Nitric oxide production in human leukocytes
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1.6.1. Isolation of human leukocytes
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Leukocytes were isolated through a controlled haemolytic shock with an ammonium
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chloride solution from buffy coats obtained from blood of healthy donors (Bossuyt et al.
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1997). The pellet was suspended in modified HBSS. Buffy coats were obtained at the
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Blood and Tissue Bank of Catalonia, under the approval of its ethical committee.
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Identity of donors was always unknown.
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1.6.2. Nitric oxide assay
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In a 96-well U bottomed microtiter plate, all experimental wells received an aliquot of
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200 μL of a suspension of human leukocytes (approximately 106 cells). Then, 20 μL of
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the different treatment dilutions or modified HBSS (negative controls) were added.
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Microtiterplates were incubated for 10 min in a stirred thermally controlled chamber at
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37 ºC. After incubation, 20 μL of LPS (3 mg/mL) and 20 µL of L-Arg (2 mg/mL) were
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added to all wells. A new incubation for 1 hour at 37 ºC and a centrifugation for 12 min
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at 2700 rpm followed. After centrifugation, aliquots of 100 μL of supernatant in each
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well were transferred to a 96-well flat bottomed microtiter plate and mixed with 100 μL
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of Griess reagent. In measuring NO stable metabolites, the colorimetric detection with
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the Griess reagent is the most commonly used technique (Griess 1879; Green et al.
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1982). After 15 min at room temperature, absorbance was read at 540 nm on a
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microtiter plate spectrophotometer, Benchmark Plus, BIORAD, USA. The amount of
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nitrite was calculated from a NaNO2 standard curve. Supernatant from leucocytes not
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exposed to LPS was used as negative control while NG-methyl-L-arginine acetate salt
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(L-NMMA), a well known inhibitor of the enzyme nitric oxide synthase (NOS) was
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used as positive control. Results were expressed as relative inhibition (%) of NO
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production. Six concentration levels of TFE were considered (17.6, 35.3, 70.5, 141.0,
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282.0 and 564.0 μg/ml). Concentration was plotted in logarithm form for both TFE and
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L-NMMA.
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1.7. Assays in animals and ethic considerations
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Female Sprague-Dawley rats (150-200 g), 8-10 weeks old were employed in the acute
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oral toxicity assay. A lineage of female outbred Syrian hamsters (50-60 g), 3-4 weeks
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old were used in the evaluation of oral mucous irritability. Animals, in a perfect health
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status, were supplied by the National Production Center for Laboratory Animals in
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Havana, Cuba (CENPALAB by its acronym in Spanish). The animals were randomly
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housed in appropriate cages at 22±3°C (on a 12 h light/dark cycle) with free access to
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water and to a standard diet (CMO 1000) supplied by CENPALAB. Animals were
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acclimated to their environment for 5 days before use for experiments. All tests
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followed the “Good Laboratory Practices” as defined by the U.S. Food and Drug
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Administration (FDA 2012). All experimental procedures using animals were in
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accordance with ethical considerations established by the Ethics Committee of the
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Toxicology and Biomedicine Centre (TOXIMED) from Santiago de Cuba Medical
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University. Research protocols were also were approved by this same committee.
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1.7.1. Acute oral toxicity evaluation
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Methodology of this assay followed the proposal of the Organization for the
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Cooperation and the Economic Development, according to the Acute Toxic Class
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Method (OECD/OCDE 423). A group of seven animals was treated with a single dose
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of 2000 mg/kg of TFE; meanwhile the control group (with the same number of animals)
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was treated with solvent. During 14 days all behaviors and physical characteristics of
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the animals were observed. Any changes in the skin or hair of the animal, color and
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appearance of mucous membranes and eyes were registered daily, as well as water and
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food consumption. Animals were weighed in days 0, 7 and 14 in a scale with a 0.01 g
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precision (Sartorius, Germany). At the end of this period animals were sacrificed by
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narcosis with an intramuscular ketamin's anesthetic dose. Anatomopathological studies
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of the digestive system's organs were performed.
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1.7.2. Oral mucous irritability
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Two groups of five animals were formed (experimental and control). A cotton pellet
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humidified with distilled water (control set) or TFE (experimental set) was placed in the
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right malar bag of each animal during 5 minutes. This procedure was repeated four
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times, once per hour. The mucous buccal appearance and the irritation grade in the
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erythema were described for every animal according to a standard reference (ISO
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10993-10). Animals were observed during 7 days after the application and weighed in
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days 0, 4 and 7. Twenty four hours later, the oral mucous of every animal was
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macroscopically examined and later they were sacrificed by cervical dislocation. For
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histological tests, tissue samples of the application site were extracted and fixed in 10%
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buffered neutral formalin for 48 hours. They were processed by paraffin embedding and
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were stained with alum-hematoxylin and eosin. Tissue sections were examined
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microscopically for histopathological change evaluation. The irritation index
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classification was developed according to the standard reference in use (ISO 10993-10).
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1.8. Statistical analysis
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All data was reported as the mean ± standard deviation (SD). For the DPPH assay
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results from six independent measurements were recorded. Relative inhibition of NO
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production was the outcome from four independent measurements. Meanwhile in the
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reducing power assay and the ferrous ion chelating activity assay results considered
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three independent experiments. Inhibitory concentration 50% (IC50) was calculated by
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interpolation in a concentration/effect curve when possible. StatGraphics plus (version
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5.0.1 for Windows, MA, USA) was used to carry out a one-way analysis of variance
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(ANOVA). When significant differences were detected by ANOVA, analyses of
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differences between the means were performed using the Tukey's HSD (Honestly
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Significant Difference Test). Two means comparison was execute by the Student's t-
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test. Values were considered significant at p < 0.05.
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